The thymus in adults infected with the HIV-1 is generally thought to be inactive, both because of age-related involution and viral destruction. We have revisited the question of thymic function in adults, using chest-computed tomography (CT) to measure thymic tissue in HIV-1-seropositive (n = 99) or HIV-1-seronegative (n = 32) subjects, and correlating these results with the level of circulating CD4(+) and CD8(+) T cells that are phenotypically described as naive thymic emigrants. Abundant thymic tissue was detectable in many (47/99) HIV-1-seropositive adults, aged 20-59. Independent of age, radiographic demonstration of thymic tissue was significantly associated with both a higher CD4(+) T cell count (P = 0.02) and a higher percentage and absolute number of circulating naive (CD45RA+CD62L+) CD4(+) T cells (P < 0.04). The prevalence of an abundant thymus was especially high in younger HIV-1-seropositive adults (= 39 yr) with CD4 counts in the range 300-500 cells/microl and in older subjects (> 40 yr) regardless of CD4 count (P = 0.03). These studies suggest that the thymus is functional in some but not all adults with HIV-1 disease.
J M McCune ... B H Gross, R M Grant
Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We used the fusion protein CTLA4Ig to block CD28-B7 T cell costimulation late after the onset of initial graft injury. Using the F334 into LEW rat model of chronic renal allograft rejection, transplant recipients were treated with a 10-d course of cyclosporine, and a subgroup received a single injection of CTLA4Ig at 8 wk after transplant. Functionally, CTLA4Ig administration prevented development of progressive proteinuria (14.3+/-4.1 mg/24 h versus 41.0+/-12.0 mg/24 h at 24 wk after transplant, P < 0.05). Histologically, graft mononuclear cell infiltration, glomerular hypertrophy, focal and segmental glomerulosclerosis, and intimal vascular hyperplasia were all attenuated in CTLA4Ig-treated animals. Lastly, reverse transcriptase-PCR and immunohistologic studies showed a significant reduction in the intragraft expression of key products of T cell and macrophage activation, and upregulation of what have recently been termed as "protective" genes, including the bcl family members, Bcl-2 and Bcl-xL, and hemoxygenase. Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection.
A Chandraker, H Azuma, K Nadeau, C B Carpenter, N L Tilney, W W Hancock, M H Sayegh
Hypoxia initiates pulmonary vasoconstriction (HPV) by inhibiting one or more voltage-gated potassium channels (Kv) in the pulmonary artery smooth muscle cells (PASMCs) of resistance arteries. The resulting membrane depolarization increases opening of voltage-gated calcium channels, raising cytosolic Ca2+ and initiating HPV. There are presently nine families of Kv channels known and pharmacological inhibitors lack the specificity to distinguish those involved in control of resting membrane potential (Em) or HPV. However, the Kv channels involved in Em and HPV have characteristic electrophysiological and pharmacological properties which suggest their molecular identity. They are slowly inactivating, delayed rectifier currents, inhibited by 4-aminopyridine (4-AP) but insensitive to charybdotoxin. Candidate Kv channels with these traits (Kv1.5 and Kv2.1) were studied. Antibodies were used to immunolocalize and functionally characterize the contribution of Kv1. 5 and Kv2.1 to PASMC electrophysiology and vascular tone. Immunoblotting confirmed the presence of Kv1.1, 1.2, 1.3, 1.5, 1.6, and 2.1, but not Kv1.4, in PASMCs. Intracellular administration of anti-Kv2.1 inhibited whole cell K+ current (IK) and depolarized Em. Anti-Kv2.1 also elevated resting tension and diminished 4-AP-induced vasoconstriction in membrane-permeabilized pulmonary artery rings. Anti-Kv1.5 inhibited IK and selectively reduced the rise in [Ca2+]i and constriction caused by hypoxia and 4-AP. However, anti-Kv1.5 neither caused depolarization nor elevated basal pulmonary artery tone. This study demonstrates that antibodies can be used to dissect the whole cell K+ currents in mammalian cells. We conclude that Kv2. 1 is an important determinant of resting Em in PASMCs from resistance arteries. Both Kv2.1 and Kv1.5 contribute to the initiation of HPV.
S L Archer ... H L Reeve, V Hampl
We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of [3H]water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice.
J D Horton, I Shimomura, M S Brown, R E Hammer, J L Goldstein, H Shimano
Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance.
A M Salmon, C Bruand, A Cardona, J P Changeux, S Berrih-Aknin
MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.
R J Riese ... H L Ploegh, H A Chapman
IFN-gamma is critical in the immune response to mycobacterial infections, and deficits in IFN-gamma production and response have been associated with disseminated nontuberculous mycobacterial infections. Mutations in the IFN-gamma receptor ligand-binding chain (IFNgammaR1) have been shown to confer susceptibility to severe infection with nontuberculous mycobacteria. However, mutations in the IFN-gamma receptor signal-transducing chain (IFNgammaR2) have not been described. We describe a child with disseminated Mycobacterium fortuitum and M. avium complex infections and absent IFN-gamma signaling due to a mutation in the extracellular domain of IFNgammaR2. In vitro cytokine production by patient PBMCs showed 75% less PHA-induced IFN-gamma production than in normal cells, while patient PHA-induced TNF-alpha production was normal. The normal augmentation of TNF-alpha production when IFN-gamma was added to endotoxin was absent from patient cells. Expression of IFNgammaR1 was normal, but there was no phosphorylation of Stat1 in response to IFN-gamma stimulation. DNA sequence analysis of the gene for IFNgammaR2 showed a homozygous dinucleotide deletion at nucleotides 278 and 279, resulting in a premature stop codon in the protein extracellular domain. This novel gene defect associated with disseminated nontuberculous mycobacterial infection emphasizes the critical role that IFN-gamma plays in host defense against mycobacteria.
S E Dorman, S M Holland
Lowering of the plasma FFA level in intact fasted rats by infusion of nicotinic acid (NA) caused essentially complete ablation of insulin secretion (IS) in response to a subsequent intravenous bolus of arginine, leucine, or glibenclamide (as previously found using glucose as the beta-cell stimulus). However, in all cases, IS became supranormal when a high FFA level was maintained by co-infusion of lard oil plus heparin. Each of these secretagogues elicited little, if any, IS from the isolated, perfused "fasted" pancreas when tested simply on the background of 3 mM glucose, but all became extremely potent when 0.5 mM palmitate was also included in the medium. Similarly, IS from the perfused pancreas, in response to depolarizing concentrations of KCl, was markedly potentiated by palmitate. As was the case with intravenous glucose administration, fed animals produced an equally robust insulin response to glibenclamide regardless of whether their low basal FFA concentration was further reduced by NA. In the fasted state, arginine-induced glucagon secretion appeared to be independent of the prevailing FFA concentration. The findings establish that the essential role of circulating FFA for glucose-stimulated IS after food deprivation also applies in the case of nonglucose secretagogues. In addition, they imply that (i) a fatty acid-derived lipid moiety, which plays a pivotal role in IS, is lost from the pancreatic beta-cell during fasting; (ii) in the fasted state, the elevated level of plasma FFA compensates for this deficit; and (iii) the lipid factor acts at a late step in the insulin secretory pathway that is common to the action of a wide variety of secretagogues.
R L Dobbins, M W Chester, B E Stevenson, M B Daniels, D T Stein, J D McGarry
Insulin resistance is instrumental in the pathogenesis of type 2 diabetes mellitus and the Insulin Resistance Syndrome. While insulin resistance involves decreased glucose transport activity in skeletal muscle, its molecular basis is unknown. Since muscle GLUT4 glucose transporter levels are normal in type 2 diabetes, we have tested the hypothesis that insulin resistance is due to impaired translocation of intracellular GLUT4 to sarcolemma. Both insulin-sensitive and insulin-resistant nondiabetic subgroups were studied, in addition to type 2 diabetic patients. Biopsies were obtained from basal and insulin-stimulated muscle, and membranes were subfractionated on discontinuous sucrose density gradients to equilibrium or under nonequilibrium conditions after a shortened centrifugation time. In equilibrium fractions from basal muscle, GLUT4 was decreased by 25-29% in both 25 and 28% sucrose density fractions and increased twofold in both the 32% sucrose fraction and bottom pellet in diabetics compared with insulin-sensitive controls, without any differences in membrane markers (phospholemman, phosphalamban, dihydropyridine-binding complex alpha-1 subunit). Thus, insulin resistance was associated with redistribution of GLUT4 to denser membrane vesicles. No effects of insulin stimulation on GLUT4 localization were observed. In non-equilibrium fractions, insulin led to small GLUT4 decrements in the 25 and 28% sucrose fractions and increased GLUT4 in the 32% sucrose fraction by 2.8-fold over basal in insulin-sensitive but only by 1.5-fold in both insulin-resistant and diabetic subgroups. The GLUT4 increments in the 32% sucrose fraction were correlated with maximal in vivo glucose disposal rates (r = +0.51, P = 0.026), and, therefore, represented GLUT4 recruitment to sarcolemma or a quantitative marker for this process. Similar to GLUT4, the insulin-regulated aminopeptidase (vp165) was redistributed to a dense membrane compartment and did not translocate in response to insulin in insulin-resistant subgroups. In conclusion, insulin alters the subcellular localization of GLUT4 vesicles in human muscle, and this effect is impaired equally in insulin-resistant subjects with and without diabetes. This translocation defect is associated with abnormal accumulation of GLUT4 in a dense membrane compartment demonstrable in basal muscle. We have previously observed a similar pattern of defects causing insulin resistance in human adipocytes. Based on these data, we propose that human insulin resistance involves a defect in GLUT4 traffic and targeting leading to accumulation in a dense membrane compartment from which insulin is unable to recruit GLUT4 to the cell surface.
W T Garvey, L Maianu, J H Zhu, G Brechtel-Hook, P Wallace, A D Baron
Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.
M H Fisher ... R A Stearns, D E MacIntyre
Fas/Fas Ligand (FasL) interactions play a significant role in peripheral T lymphocyte homeostasis and in certain pathological states characterized by T cell depletion. In this study, we demonstrate that antigen-presenting cells such as monocyte-derived human macrophages (MDM) but not monocyte-derived dendritic cells express basal levels of FasL. HIV infection of MDM increases FasL protein expression independent of posttranslational mechanisms, thus highlighting the virus-induced transcriptional upregulation of FasL. The in vitro relevance of these observations is confirmed in human lymphoid tissue. FasL protein expression is constitutive and restricted to tissue macrophages and not dendritic cells. Moreover, a significant increase in macrophage-associated FasL is observed in lymphoid tissue from HIV (+) individuals (P < 0.001), which is further supported by increased levels of FasL mRNA using in situ hybridization. The degree of FasL protein expression in vivo correlates with the degree of tissue apoptosis (r = 0.761, P < 0. 001), which is significantly increased in tissue from HIV-infected patients (P < 0.001). These results identify human tissue macrophages as a relevant source for FasL expression in vitro and in vivo and highlight the potential role of FasL expression in the immunopathogenesis of HIV infection.
D H Dockrell ... P J Leibson, C V Paya
In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.
S W Dow, R E Elmslie, A P Willson, L Roche, C Gorman, T A Potter
Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology characterized by progressive biventricular failure, cardiac dilatation, and premature mortality. Here we show that transgenic mice expressing a dominant-negative form of the CREB transcription factor (CREBA133) under the control of the cardiac myocyte-specific alpha-MHC promoter develop dilated cardiomyopathy that closely resembles many of the anatomical, physiological, and clinical features of human IDC. Between 2 and 20 wk of age, these mice develop four chamber cardiac dilatation, decreased systolic and diastolic left ventricular function, and attenuated contractile responses to the beta-adrenergic agonist, isoproterenol. Histologically, the CREBA133 hearts demonstrated both atrophic and hypertrophied fibers as well as significant interstitial fibrosis. These anatomical and hemodynamic changes were associated with hepatic congestion and peripheral edema, intracardiac thrombi, and premature mortality. Taken together, these results implicate CREB as an important regulator of cardiac myocyte function and provide a genetic model of dilated cardiomyopathy which should facilitate studies of both the pathogenesis and therapy of this clinically important disorder.
R C Fentzke, C E Korcarz, R M Lang, H Lin, J M Leiden
The binding of beta2 (CD18) integrins on PMN cell membrane to intercellular adhesion molecule (ICAM) counter-receptors on the surface of vascular endothelial cells mediates PMN adhesion to endothelial cells. Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm (Ancylostoma caninum), is a beta2 integrin antagonist that inhibits PMN adhesion to endothelial cells. We transferred the NIF gene into CD1 mouse lungs by intravenous injection of cationic liposomes to study the effects of in vivo NIF expression on LPS-induced lung PMN sequestration and the development of lung injury. RT-PCR and Northern blot analysis indicated the lung-selective expression of the NIF transgene, and immunocytochemistry showed prominent NIF expression in pulmonary microvessel endothelial cells. NIF staining was also observed in intraluminal leukocytes present in pulmonary microvessels. This may be the result of NIF binding to leukocytes after its secretion from the transduced lung cells, since there was no evidence of NIF gene expression in circulating leukocytes. Pulmonary vascular NIF expression abrogated the lung tissue PMN uptake and airspace migration of PMN and prevented lung vascular injury (as measured by the lung tissue uptake of [125I]labeled albumin) after the intraperitoneal LPS challenge (200 microg/mouse). Expression of a control protein, chloramphenicol acetyltransferase (CAT), by the same strategy, had no effect on these responses. In vitro studies showed that NIF prevented mouse PMN adhesion consistent with the inhibition of lung uptake after LPS challenge in NIF transgene-expressing mice. We conclude that pulmonary vascular expression of NIF, a specific beta2 integrin- binding protein, is a potentially useful gene transfer strategy in modulating the infiltration of PMN across the alveolar-capillary epithelial barrier and in preventing lung vascular endothelial injury.
M Y Zhou, S K Lo, M Bergenfeldt, C Tiruppathi, A Jaffe, N Xu, A B Malik
Leukocyte Adhesion Deficiency Type II (LAD II) is a recently described syndrome and the two patients with this defect lack fucosylated glycoconjugates. These glycoconjugates include the selectin ligand, sialyl LewisX, and various fucosylated blood group antigens. To date, the molecular anomaly in these patients has not been identified. We localized the defect in LAD II to the de novo pathway of GDP-fucose biosynthesis, by inducing cell-surface expression of fucosylated glycoconjugates after exposure of lymphoblastoid cell lines from the LAD II patients to exogenous fucose. This defect is not restricted to hematopoietic cells, since similar findings were elicited in both human umbilical vein endothelial cells (HUVEC) and fibroblasts derived from an affected abortus. We have used these LAD II endothelial cells to examine the consequence of fucosylation of endothelial cells on the rolling of normal neutrophils in an in vitro assay. Neutrophil rolling on LPS-treated normal and LAD II HUVEC was inhibited by an E-selectin monoclonal antibody at both high and low shear rates. LAD II HUVEC lacking fucosylated glycoproteins supported leukocyte rolling to a similar degree as normal HUVEC or LAD II cells that were fucose-fed. At low shear rates, an L-selectin antibody inhibited neutrophil rolling to a similar degree whether the LAD II cells had been fucose-fed or not. These findings suggest that fucosylation of nonlymphoid endothelial cells does not play a major role in neutrophil rolling and that fucose is not a critical moiety on the L-selectin ligand(s) on endothelial cells of the systemic vasculature.
A Karsan ... H D Ochs, J M Harlan
Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and anti-CD154 (anti-CD40-ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 d without further intervention. The protocol induces long-term allograft survival, but the mechanism is unknown. We now report: (a) addition of thymectomy to the protocol permitted skin allografts to survive for > 100 d, suggesting that graft rejection in euthymic mice results from thymic export of alloreactive T cells. (b) Clonal deletion is not the mechanism of underlying long-term graft survival, as recipient thymectomized mice were immunocompetent and harbor alloreactive T cells. (c) Induction of skin allograft acceptance initially depended on the presence of IFN-gamma, CTLA4, and CD4(+) T cells. Addition of anti-CTLA4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect. The role of IFN-gamma was confirmed using knockout mice. (d) Graft survival was associated with the absence of IFN-gamma in the graft. (e) Long-term graft maintenance required the continued presence of CD4(+) T cells. The results suggest that, with modification, our short-term protocol may yield a procedure for the induction of long-term graft survival without prolonged immunosuppression.
T G Markees, N E Phillips, E J Gordon, R J Noelle, L D Shultz, J P Mordes, D L Greiner, A A Rossini
Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.
F Prosper, D Stroncek, J B McCarthy, C M Verfaillie
Chronic hypoxia induces pulmonary hypertension and right ventricular (RV) hypertrophy. Nitric oxide (NO) has been proposed to modulate the pulmonary vascular response to hypoxia. We investigated the effects of congenital deficiency of endothelial NO synthase (NOS3) on the pulmonary vascular responses to breathing 11% oxygen for 3-6 wk. After 3 wk of hypoxia, RV systolic pressure was greater in NOS3-deficient than in wild-type mice (35+/-2 vs 28+/-1 mmHg, x+/-SE, P < 0.001). Pulmonary artery pressure (PPA) and incremental total pulmonary vascular resistance (RPI) were greater in NOS3-deficient than in wild-type mice (PPA 22+/-1 vs 19+/-1 mmHg, P < 0.05 and RPI 92+/-11 vs 55+/-5 mmHg.min.gram.ml-1, P < 0.05). Morphometry revealed that the proportion of muscularized small pulmonary vessels was almost fourfold greater in NOS3-deficient mice than in wild-type mice. After 6 wk of hypoxia, the increase of RV free wall thickness, measured by transesophageal echocardiography, and of RV weight/body weight ratio were more marked in NOS3-deficient mice than in wild-type mice (RV wall thickness 0.67+/-0.05 vs 0.48+/-0.02 mm, P < 0.01 and RV weight/body weight ratio 2.1+/-0.2 vs 1.6+/-0.1 mg. gram-1, P < 0.05). RV hypertrophy produced by chronic hypoxia was prevented by breathing 20 parts per million NO in both genotypes of mice. These results suggest that congenital NOS3 deficiency enhances hypoxic pulmonary vascular remodeling and hypertension, and RV hypertrophy, and that NO production by NOS3 is vital to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress.
W Steudel, M Scherrer-Crosbie, K D Bloch, J Weimann, P L Huang, R C Jones, M H Picard, W M Zapol
Anterior pituitary hormone secretion is mainly regulated by hypothalamic releasing factors, which reach the pituitary via portal vessels. It has been demonstrated recently that these peptides can also be produced by the pituitary itself, thus possibly modulating hormone secretion in a paracrine/autocrine fashion. The object of this study was to seek evidence for the synthesis and secretion of corticotropin-releasing hormone (CRH) within the anterior pituitary and to ascertain its biological relevance. Messenger RNA from adult rat anterior pituitary fragments and cell cultures was reverse transcribed and subjected to PCR amplification using primers specific to the rat CRH gene. As in the hypothalamus, a single 232-bp band was obtained. The correspondence of the amplified fragment to the sequence of the CRH gene was confirmed by Southern blotting and restriction enzyme digestion. Combined in situ reverse transcription-PCR amplification/immunocytochemistry demonstrated the presence of CRH mRNA in corticotropes. Medium from anterior pituitary primary cultures contained approximately 7 pg/microg protein of CRH immunoreactivity which presented the same chromatographic profile on HPLC as the mature CRH peptide. Incubation of anterior pituitary cells with an antibody directed against CRH markedly reduced basal ACTH secretion compared with serum-treated control wells (0.89+/-0.11 vs. 1.74+/-0.14 ng/200,000 cells in control wells after 1 h, P < 0.05; 1.17+/-0.10 vs. 2.16+/-0. 39 ng/200,000 cells after 2 h, P < 0.05; 1.45+/-0.12 vs. 3.12+/-0.61 ng/200,000 cells after 3 h, P < 0.05). Further, the ACTH response to potassium and to forskolin was markedly blunted by the CRH antiserum as well as by the CRH antagonist, alpha-helical CRH(9-41). In conclusion, this study demonstrates the presence of CRH mRNA in normal rat corticotropes and the secretion of the mature peptide by the anterior pituitary, pointing to the production of CRH at the site of its target cells. In addition, intrapituitary CRH contributes in a paracrine/autocrine fashion to ACTH secretion.
F P Giraldi, F Cavagnini
We analyzed the inheritance of body fat, leptin levels, plasma lipoprotein levels, insulin levels, and related traits in an intercross between inbred mouse strains CAST/Ei and C57BL/6J. CAST/Ei mice are unusually lean, with only approximately 8% of body weight as fat, whereas C57BL/6J mice have approximately 18% body fat. Quantitative trait locus analysis using > 200 F2 mice revealed highly significant loci (lod scores > 4.3) on chromosomes 2 (three separate loci) and 9 that contribute to mouse fat-pad mass for mice on a high-fat diet. Some loci also influenced plasma lipoprotein levels and insulin levels either on chow or high-fat diets. Two loci for body fat and lipoprotein levels (on central and distal chromosome 2) coincided with a locus having strong effects on hepatic lipase activity, an activity associated with visceral obesity and lipoprotein levels in humans. A locus contributing to plasma leptin levels (lod score 5.3) but not obesity was identified on chromosome 4, near the leptin receptor gene. These data identify candidate regions and candidate genes for studies of human obesity and diabetes, and suggest obesity is highly complex in terms of the number of genetic factors involved. Finally, they support the existence of specific genetic interactions between body fat, insulin metabolism, and lipoprotein metabolism.
M Mehrabian, P Z Wen, J Fisler, R C Davis, A J Lusis
Inhaled nitric oxide (NO) is being used more and more in intensive care units as a modality to improve the outcome of patients with pulmonary complications. Our objective was to demonstrate that inhaled NO could impact upon a distally inflamed microvasculature-improving perfusion, leukocyte adhesive interactions, and endothelial dysfunction. Using intravital microscopy to visualize ischemia/reperfusion of postcapillary venules, we were able to demonstrate that the reduction in perfusion, the dramatic increase in leukocyte rolling, adhesion, and emigration, and the endothelial dysfunction could all be significantly abrogated with 80 ppm, but not 20 ppm inhaled NO. Perfusing whole blood directly over an inert P-selectin and CD18 ligand substratum incorporated in a flow chamber recruited the same number of rolling and adhering leukocytes from NO-ventilated and non-NO-ventilated animals, suggesting that inhaled NO was not directly affecting leukocytes. To demonstrate that inhaled NO was actually reaching the peripheral microvasculature in vivo, we applied a NO synthase inhibitor locally to the feline mesentery and demonstrated that the vasoconstriction, as well as leukocyte recruitment, were essentially abolished by inhaled NO, suggesting that a NO-depleted peripheral microvasculature could be replenished with inhaled NO in vivo. Finally, inhaled NO at the same concentration that was effective in ischemia/reperfusion did not affect vascular alterations, leukocyte recruitment, and endothelial dysfunction associated with endotoxemia in the feline mesentery. In conclusion, our data for the first time demonstrate a role for inhaled NO as a therapeutic delivery system to the peripheral microvasculature, showing tremendous efficacy as an antiadhesive, antivasoconstrictive, and antipermeabilizing molecule in NO-depleted tissues, but not normal microvessels or vessels that have an abundance of NO (LPS-treated). The notion that blood borne molecules have NO carrying capacity is conceptually consistent with our observations.
A Fox-Robichaud, D Payne, S U Hasan, L Ostrovsky, T Fairhead, P Reinhardt, P Kubes
A massive infiltration of the skin by activated CD8+ T lymphocytes involving both the dermis and the epidermis has been found in HIV-1-infected patients presenting with a chronic skin rash. We characterized the T cell receptor (TCR) BV-BJ junctional diversity of the skin-infiltrating lymphocytes (SILs) in four patients. The SILs expressed a limited set of TCRBV gene segments. Complementarity determining region 3 length analysis further emphasized their oligoclonality, suggesting that antigen stimulation might be responsible for the cutaneous T cell expansion. Furthermore, independent skin biopsies obtained from the same individual were shown to harbor distinct T cell repertoires, possibly reflecting the spatial heterogeneity of the antigenic stimuli. The CD8+ cytotoxic T lymphocyte (CTL) lines isolated from the skin rash in one patient exhibited a specific, class I MHC-restricted cytotoxic activity against HIV-1 Gag- and Pol-expressing target cells, whereas CTL lines derived from the skin lesions of a second patient were shown to be predominantly Env-specific. Taken together, these data demonstrate the infiltration of HIV-specific CTLs in the skin of HIV-infected patients, and suggest that in addition to their known role in controlling the retroviral infection, these CTLs may also be involved in the pathogenesis of cutaneous inflammatory disorders occurring during the course of HIV infection.
H Bachelez, F Hadida, C Parizot, B Flageul, M Kemula, L Dubertret, P Debree, G Gorochov
Allospecific CD8(+) T lymphocytes are an important component of the cellular response in allograft rejection. These cells recognize and engage MHC class I antigens, leading to allospecific cytolytic responses and graft rejection. In mouse kidney allografts that survive to 3 wk after transplantation, we noted that the majority of CD8(+) cells do not express surface alpha/beta T cell receptor alpha/beta(TCR), gamma/deltaTCR, or CD3. However, these CD8(+)TCR- cells did express surface markers characteristic of T cells, including Thy1.2, CD2, and CD5. In addition, the CD8(+)TCR- cells expressed mRNA for TCR Vbeta gene families, and nearly half stained positive for cytoplasmic Vbeta8 protein, suggesting that they are T cells that have downregulated alpha/betaTCR protein expression from their cell surfaces. When these surface TCR- cells were isolated from kidney allografts by flow cytometry and cultured in the presence of either allogeneic or syngeneic stimulators, nearly 100% of cells reacquired normal levels of alpha/betaTCR expression with disproportionate usage of Vbeta8 chains. After recovery of their surface TCR expression, the CD8(+)TCR- population demonstrated strong alloreactivity in culture. These results suggest that the substantial number of CD8(+)TCR- cells found in long-term surviving mouse kidney allografts are alpha/beta-T cells that have downregulated their cell surface expression of TCR. While in other systems this phenotype may identify cells that have engaged antigen, our results indicate that loss of TCR expression by CD8(+) kidney graft-infiltrating cells may not depend on antigen engagement and that elements in the microenvironment of the kidney graft play a key role in this process. Factors that modulate expression of TCR by graft-infiltrating lymphocytes may have an important role in regulating rejection responses.
R B Mannon, B L Kotzin, C Nataraj, K Ferri, E Roper, R J Kurlander, T M Coffman
Impairment of insulin secretion due to prolonged hyperglycemia is believed to contribute to the manifestation of diabetes mellitus, often referred to as glucose toxicity of pancreatic beta cells. In addition, impaired beta cell function has been associated with elevated islet triglyceride content (lipotoxicity). Impaired functions of the transactivating factors islet duodenum homeobox-1 (IDX-1) and RIPE3b-binding proteins have been implicated in the pathological downregulation of insulin gene transcription by high glucose levels in pancreatic beta cell lines in vitro, and, similarly, the exposure of pancreatic islets to fatty acids decreases IDX-1 expression. Previously, we identified the basic leucine zipper transcription factor CCAAT/enhancer-binding protein beta (C/ EBPbeta) as an inhibitor of insulin gene transcription in pancreatic beta cells and showed that the expression of C/EBPbeta is upregulated in insulinoma-derived beta cell lines by sustained high glucose concentrations. Here we describe the regulation of the expression of IDX-1, C/EBPbeta, and insulin at the mRNA and protein levels in pancreatic islets in animal models of diabetes mellitus. Concomitant with a downregulation of IDX-1 and insulin expression, C/EBPbeta is upregulated in association with the manifestation of hyperglycemia during the development of diabetes in the Zucker diabetic fatty (fa/fa) rat and in the 90% pancreatectomy rat model of diabetes. This regulation is demonstrated to influence both the amount of cellular protein and the level of steady state messenger RNA. Our findings indicate that the differential dysregulation of both IDX-1 and C/EBPbeta, in response to sustained hyperglycemia or hyperlipidemia, may be involved in the impairment of insulin gene expression during the manifestation of diabetes mellitus.
J Seufert, G C Weir, J F Habener
The primary response transcription factor, early growth response-1 (Egr-1), is rapidly activated by a variety of extracellular stimuli. Egr-1 binds to a sequence found in the promoters of genes involved in vascular injury, such as PDGF-A and tissue factor, and trans-activates their expression in endothelial cells in response to fluid shear stress. Here we show that egr-1 mRNA is increased after 30 min of flow in human aortic endothelial cell and HeLa cell cultures. Transient transfection of HeLa cells with reporter gene constructs driven by the murine or human egr-1 5' flanking sequence revealed a five- and ninefold induction, respectively, in transcriptional activity after exposure to a shear stress of 5 dynes/cm2 for 3 h. Deletion of sequences in the murine promoter containing two AP1 sites and an inhibitory Egr-1 binding sequence, did not reduce shear stress inducibility. However, progressive deletion of five serum response elements, reduced both the basal promoter activity and its capacity to be activated by shear stress. Further examination indicated that the three upstream serum response elements are predominantly responsible for shear stress activation of the egr-1 promoter. Treatment of cells with PD98059, a specific inhibitor of mitogen-activated protein kinase-1 inhibited shear stress activation of egr-1. We suggest that egr-1 activation by shear stress involves activation of Elk-1 but not c-jun activity. These data, which are consistent with previous findings for shear mediated signaling via the mitogen-activated protein kinase cascade, now implicate shear modulation of the Egr-1 transcription factor in this pathway.
J L Schwachtgen, P Houston, C Campbell, V Sukhatme, M Braddock
Sickle red cells bind significant amounts of soluble laminin, whereas normal red cells do not. Solid phase assays demonstrate that B-CAM/LU binds laminin on intact sickle red cells and that red cell B-CAM/LU binds immobilized laminin, whereas another putative laminin binding protein, CD44, does not. Ligand blots also identify B-CAM/LU as the only erythrocyte membrane protein(s) that binds laminin. Finally, transfection of murine erythroleukemia cells with human B-CAM cDNA induces binding of both soluble and immobilized laminin. Thus, B-CAM/LU appears to be the major laminin-binding protein of sickle red cells. Previously reported overexpression of B-CAM/LU by epithelial cancer cells suggests that this protein may also serve as a laminin receptor in malignant tumors.
M Udani, Q Zen, M Cottman, N Leonard, S Jefferson, C Daymont, G Truskey, M J Telen
Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.
C Brander ... B D Walker, S A Kalams
We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.
T Murohara ... P L Huang, J M Isner
We have recently reported that tumor-associated lymphocytes obtained from ascitic fluids of women with ovarian carcinoma (OvCA) demonstrate a marked decrease in expression of cytoplasmic CD3-zeta and surface CD3-epsilon chains, which is associated with altered function of T cell receptor (TcR). We now demonstrate that OvCAs in situ and in culture express functional Fas ligand (FasL), capable of triggering an intrinsic cell death program in Fas-expressing T cells. The possibility of a relationship between cell death and altered expression of TcR was examined. The data indicate that alterations in expression of CD3-zeta and CD3-epsilon chains in T cells coincubated with OvCA are related to tumor-induced apoptosis, as the addition of pan-caspase inhibitors, DEVD-cho or YVAD-cho, prevents both the in vitro induction of T cell death by OvCA cells and the changes in the level of expression of CD3-zeta and CD3-epsilon chains. In the presence of Fas-Fc fusion protein, but not Fc-control protein, the loss in expression of CD3-zeta and CD3-epsilon chains induced in T cells by FasL+ OvCA cells was prevented. These results suggest that the loss in expression of CD3-zeta and CD3-epsilon chains in T lymphocytes interacting with OvCA cells is associated with apoptosis mediated by FasL-expressing tumor cells.
H Rabinowich, T E Reichert, Y Kashii, B R Gastman, M C Bell, T L Whiteside
The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.
Y Maeshima ... Y S Kanwar, H Makino
PMN-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease. Epithelial cells respond to organisms such as Pseudomonas aeruginosa, the major pathogen in CF, by expressing the leukocyte chemokine IL-8. Experiments were performed using several different types of respiratory epithelial cells that demonstrate that ligation of ceramide-associated receptors on epithelial surfaces by P. aeruginosa pili is a major stimulus for the translocation of transcription factor nuclear factor (NF)-kappaB and initiation of IL-8 expression by epithelial cells. Using electrophoretic mobility shift assays and Western hybridizations, nuclear NF-kappaB was found shortly after epithelial cells were stimulated by either whole organisms, isolated pili, or antibody to the pilin receptor asialoGM1. IB3 cells, which express mutations in cystic fibrosis transmembrane conductance regulator (CFTR) (DeltaF508/W1282X), were noted to have significantly greater amounts of endogenous nuclear NF-kappaB, but not the transcription factor C/EBP, than CF cells corrected by episomal copies of normal CFTR (C-38) or IB3 cells grown at a permissive temperature (25 degreesC). Activation of NF-kappaB and subsequent IL-8 expression in epithelial cells can result from activation of at least two pathways: an exogenous signaling cascade that is activated by ligation of ceramide-associated adhesins such as P. aeruginosa pilin, or endogenous stimulation, suggested to be a consequence of cell stress caused by the accumulation of mutant CFTR in the endoplasmic reticulum.
E DiMango, A J Ratner, R Bryan, S Tabibi, A Prince
A Lomri, R Evers, C Mohan, G Teoh, HS Taylor, E Kim, L Liaw
K Asano, H Nakamura, C M Lilly, M Klagsbrun, J M Drazen
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